High frequency somatic embryogenesis and plant regeneration in root explant cultures of carnation

Root explants excised from carnation plants maintained in vitro formed off-white, friable calluses after three weeks of culture on Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ thidiazuron (TDZ) and 1 mg l⁻¹ α-naphthalaneacetic acid (NAA). These calluses were subsequently transferred to MS basal medium where, after an additional four weeks of culture, approximately 50% of the calluses formed somatic embryos. However, calluses formed on root explants that had been cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid did not produce somatic embryos upon transfer to MS basal medium. Somatic embryos developed into plantlets and subsequently were grown to maturity. These results indicate that root explants have a high competence for somatic embryogenesis in carnation. J. Seo and S.W. Kim contributed equally to this work.     

Relationships Between Catecholamine Levels and Stress or Intelligence

Catecholamines, including epinephrine (E), norepinephrine (NE), and dopamine (DA), are associated with the response to stressful conditions. However, the relationships of catecholamines with intelligence and their interactions with stress remain unclear. This study assessed stress, intelligence quotient (IQ), and catecholamine levels in 70 healthy subjects to elucidate associations between catecholamines and stress, and between catecholamines and IQ. Additionally, the associations of catecholamines with stress and IQ were analyzed according to hemispheric dominance using the Brain Preference Indicator (BPI). There were positive correlations between the NE/E ratio and the somatization of stress but negative correlations between the E/NE ratio and the somatization of stress among the total number of subjects. In the right-brain-dominant group, a high E/DA ratio was correlated with low levels of stress, somatization and depression, and high NE/E and DA/E ratios were associated with high levels of somatization. In the left-brain-dominant group, high E levels were correlated with low levels of depression. In the total subjects, there were positive correlations between the NE/E and DA/E ratios and the sum of the vocabulary, arithmetic, picture arrangement, and block design IQ subtests. Thus, these catecholamines were associated with stress and IQ, which suggests that the autonomic functional regulation of catecholamine levels in relation to stress may also affect cognitive functions related to intelligence in the brain. Furthermore, the relationships between catecholamines and stress or IQ differed depending on hemispheric dominance, which suggests that the present results could be used to inform the development of personalized therapies based on hemispheric asymmetry.

     

Effects of Radix Polygalae on Cognitive Decline and Depression in Estradiol Depletion Mouse Model of Menopause

Postmenopausal syndrome refers to symptoms caused by the gradual decrease in female hormones after mid-40 years. As a target organ of estrogen, decrease in estrogen causes various changes in brain function such as a decrease in choline acetyltransferase and brain-derived neurotrophic factor; thus, postmenopausal women experience cognitive decline and more depressive symptoms than age-matched men. Radix Polygalae has been used for memory boosting and as a mood stabilizer and its components have shown neuroprotective, antidepressant, and stress relief properties. In a mouse model of estrogen depletion induced by 4-vinylcyclohexene diepoxide, Radix Polygalae was orally administered for 3 weeks. In these animals, cognitive and depression-related behaviors and molecular changes related to these behaviors were measured in the prefrontal cortex and hippocampus. Radix Polygalae improved working memory and contextual memory and despair-related behaviors in 4-vinylcyclohexene diepoxide-treated mice without increasing serum estradiol levels in this model. In relation to these behaviors, choline acetyltransferase and brain-derived neurotrophic factor in the prefrontal cortex and hippocampus and bcl-2-associated athanogene expression increased in the hippocampus. These results implicate the possible benefit of Radix Polygalae in use as a supplement of estrogen to prevent conditions such as postmenopausal depression and cognitive decline.     

Production of a hepatoprotective cerebroside from suspension cultures of Lycium chinense

Suspension cultures derived from Lycium chinense Miller seedlings produced significant amounts of a hepatoprotective cerebroside. Callus was induced from the stem of aseptic seedlings of L. chinense and maintained on MS solid media supplemented with 1.0 ppm 2,4-D and 0.1 ppm kinetin. Suspension cultures were established, and the cells were grown in the same liquid media in the dark. Lyophilized cells were extracted with a combined reagent of chloroform and methanol (2:1, v/v). An aqueous suspension of the evaporated cell extract was partitioned with chloroform, and the chloroform layer was subjected to silicic acid column chromatography followed by semi-preparative reverse phase C8 high pressure liquid chromatography. The purified compound showed hepatoprotective activity comparable to that shown by silymarin, and the structure was identified as 1-O-(β-d-glucopyranosyl)-(2S,3R,4E,8Z)-2-N-2′-hydroxy-(palmitoyl)-4,8-sphingadiene on the basis of spectral data. The content of the compound in cultured cell was tenfold higher than that of the fruit of L. chinense. The biosynthesis of the compound in cultured cell systems appears to parallel cell growth. Received: 12 June 1998 / Revision received: 30 July 1998 / Accepted: 14 August 1998     

Characterization and clinical manifestations of Arcanobacterium phocae infections in marine mammals stranded along the central California coast

Between 1994 and 2000, 141 Arcanobacterium phocae isolates were recovered from marine mammals that stranded along the central California coast (USA). Arcanobacterium phocae was cultured from tissue sites with abnormal discharge or evidence of inflammation in 66 California sea lions (Zalophus californianus), 50 Pacific harbor seals (Phoca vitulina richardii), 19 northern elephant seals (Mirounga angustirostris), five southern sea otters (Enhydra lutris nereis), and one common dolphin (Delphinus delphis). The overall prevalence of A. phocae among cultured stranded marine mammals was 8%. This is the first report of A. phocae in animals from the Pacific Ocean. Sequence analysis of a portion of the 16S ribosomal RNA gene confirmed recent isolates as A. phocae. Prior to phylogenetic testing and the routine use of the esculin hydrolysis and motility tests, A. phocae isolates may have been misidentified as Listeria ivanovii. Arcanobacterium phocae was commonly isolated from superficial abscesses, was often present in mixed infections, and was susceptible to all antimicrobial agents tested.     

Production of transgenic canine embryos using interspecies somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry 'foreign' DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6?. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8-16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8-16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine-bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models.     

SIRT1 and c-Myc Promote Liver Tumor Cell Survival and Predict Poor Survival of Human Hepatocellular Carcinomas

The increased expression of SIRT1 has recently been identified in numerous human tumors and a possible correlation with c-Myc oncogene has been proposed. However, it remains unclear whether SIRT1 functions as an oncogene or tumor suppressor. We sought to elucidate the role of SIRT1 in liver cancer under the influence of c-Myc and to determine the prognostic significance of SIRT1 and c-Myc expression in human hepatocellular carcinoma. The effect of either over-expression or knock down of SIRT1 on cell proliferation and survival was evaluated in both mouse and human liver cancer cells. Nicotinamide, an inhibitor of SIRT1, was also evaluated for its effects on liver tumorigenesis. The prognostic significance of the immunohistochemical detection of SIRT1 and c-Myc was evaluated in 154 hepatocellular carcinoma patients. SIRT1 and c-Myc regulate each other via a positive feedback loop and act synergistically to promote hepatocellular proliferation in both mice and human liver tumor cells. Tumor growth was significantly inhibited by nicotinamide in vivo and in vitro. In human hepatocellular carcinoma, SIRT1 expression positively correlated with c-Myc, Ki67 and p53 expression, as well as high á-fetoprotein level. Moreover, the expression of SIRT1, c-Myc and p53 were independent prognostic indicators of hepatocellular carcinoma. In conclusion, this study demonstrates that SIRT1 expression supports liver tumorigenesis and is closely correlated with oncogenic c-MYC expression. In addition, both SIRT1 and c-Myc may be useful prognostic indicators of hepatocellular carcinoma and SIRT1 targeted therapy may be beneficial in the treatment of hepatocellular carcinoma.     

The Balance of Apoptotic and Necrotic Cell Death in Mycobacterium tuberculosis Infected Macrophages Is Not Dependent on Bacterial Virulence

Background

An important mechanism of Mycobacterium tuberculosis pathogenesis is the ability to control cell death pathways in infected macrophages: apoptotic cell death is bactericidal, whereas necrotic cell death may facilitate bacterial dissemination and transmission.

Methods

We examine M.tuberculosis control of spontaneous and chemically induced macrophage cell death using automated confocal fluorescence microscopy, image analysis, flow cytometry, plate-reader based vitality assays, and M.tuberculosis strains including H37Rv, and isogenic virulent and avirulent strains of the Beijing lineage isolate GC1237.

Results

We show that bacterial virulence influences the dynamics of caspase activation and the total level of cytotoxicity. We show that the powerful ability of M.tuberculosis to inhibit exogenously stimulated apoptosis is abrogated by loss of virulence. However, loss of virulence did not influence the balance of macrophage apoptosis and necrosis – both virulent and avirulent isogenic strains of GC1237 induced predominantly necrotic cell death compared to H37Rv which induced a higher relative level of apoptosis.

Conclusions

This reveals that macrophage necrosis and apoptosis are independently regulated during M. tuberculosis infection of macrophages. Virulence affects the level of host cell death and ability to inhibit apoptosis but other strain-specific characteristics influence the ultimate mode of host cell death and alter the balance of apoptosis and necrosis.